Dyeing agent having at least two dyes for staining a biological sample and staining method employing the dyeing agent

Abstract

Dyeing agents which are excellent in visual recognition of discernible or tangible components in a sample cause no coagulation of proteins, sugars or glycoproteins dissolved in the sample. An apparatus for image analysis of flow type stain particles uses the dyeing agents, and particles of discernible components suspended in a flowing sample can be detected so that images of the particles can be efficiently photographed and the discernible components can thus be analyzed by image processing of the thus-obtained image.

What is claimed is: 
     
       1. A dyeing agent for staining a biological sample, which comprises an azo dye and a xanthene dye suitable for supravital staining of cells and tissue, and a pH buffer, said azo and xanthene dyes being nonpolar molecular dyes which, upon blending, cause no agglutination or sedimentation of the dyes and which do not precipitate or agglutinate sugars, proteins or glycoproteins dissolved in said biological sample; wherein the blended azo and xanthene dyes are suitable for staining at least two objects, to different respective degrees in color hue or in dyeing strength depending on the respective objects; and wherein the blended azo and xanthene dyes are suitable for staining respective components of a single object to different degrees in color hue or in dyeing strength.   
     
     
       2. A dyeing agent according to claim 1, wherein said azo dye is selected from the group consisting of Evans Blue and Trypan Blue. 
     
     
       3. A dyeing agent according to claim 1, wherein said xanthene dye is selected from the group consisting of Erythrosine, Phloxine and Eosin. 
     
     
       4. A dyeing agent according to claim 2, wherein said xanthene dye is selected from the group consisting of Erythrosine, Phloxine and Eosin. 
     
     
       5. A dyeing agent according to claim 4, wherein said pH buffer is selected from the group consisting of a phosphate buffer, a succinate buffer and a tris-acid buffer. 
     
     
       6. A dyeing agent according to claim 5, further comprising an antibacterial agent as a fixative agent. 
     
     
       7. A dyeing agent according to claim 6, wherein said antibacterial agent is selected from the group consisting of sodium azide, para-hydroxyphenylacetic acid, dehydroacetic acid and ethylenediaminetetraacetic acid. 
     
     
       8. A dyeing agent obtained by mixing one volume of about 0.2 to 10.0×10 -2  mol/l of Evans Blue or Trypan Blue with about 0.5 to 2.0 volumes of about 0.2 to 10.0×10 -2  mol/l of Erythrosine; and adjusting a pH of the system to 5.7 to 7.9 using 1/30 to 1/5 mols/l of a solvent selected from the group consisting of a phosphate buffer, a succinate buffer and a tris-acid buffer. 
     
     
       9. A dyeing agent according to claim 1, further comprising a fixative agent. 
     
     
       10. A dyeing agent according to claim 9 obtained by mixing one volume of about 0.2 to 10.0×10 -2  mol/l of Evans Blue or Trypan Blue as the azo dye with about 0.5 to 2.0 volumes of about 0.2 to 10.0×10 -2  mol/l of Phloxine as the xanthene dye; adjusting a pH of the system to 5.7 to 7.9 using 1/30 to 1/5 mols/l of a solvent selected from the group consisting of a phosphate buffer, a succinate buffer and a tris-acid buffer; and further adding an agent selected from the group consisting of glutaraldehyde, formaldehyde and paraformaldehyde as the fixative agent to the mixture in a concentration of 0.02 to 5.0%. 
     
     
       11. A dyeing agent according to claim 9, further comprising a surface active agent. 
     
     
       12. A dyeing agent according to claim 11, which is obtained by mixing one volume of about 0.2 to 10.0×10  -2  mol/l of Evans Blue or Trypan Blue as the azo dye with about 0.5 to 2.0 volumes of about 0.2 to 10.0×10 -2  mol/l of Eosine as the xanthene dye; adjusting a pH of the system to 5.7 to 7.9 using 1/30 to 1/5 mols/l of a solvent selected from the group consisting of a phosphate buffer, a succinate buffer and a tris-acid buffer; and further adding an agent selected from the group consisting of glutaraldehyde, formaldehyde and paraformaldehyde as the fixative agent to the mixture in a concentration of 0.02 to 5.0%, and sodium dodecyl sulfate as the surface active agent in a concentration of 0.01 to 0.5%. 
     
     
       13. A method for staining discernible urinary sediments in urine, comprising the step of using a dyeing agent according to claim 7. 
     
     
       14. A dyeing agent according to claim 8, wherein the dyeing agent is further obtained by mixing with the mixture one member selected from the group consisting of sodium azide, parahydroxyphenylacetic acid, dehydroacetic acid and ethylenediaminetetraacetic acid in a concentration of 0.01 to 1.0%. 
     
     
       15. A dyeing agent according to claim 10, wherein the dyeing agent is further obtained by mixing with the mixture one member selected from the group consisting of sodium azide, para-hydroxyphenylacetic acid, dehydroacetic acid and ethylenediaminetetraacetic acid in a concentration of 0.01 to 1.0%. 
     
     
       16. A dyeing agent according to claim 12, wherein the dyeing agent is further obtained by mixing with the mixture one member selected from the group consisting of sodium azide, para-hydroxyphenylacetic acid, dehydroacetic acid and ethylenediaminetetraacetic acid in a concentration of 0.01 to 1.0%. 
     
     
       17. A dyeing agent for staining a biological sample, obtained by mixing one volume of about 0.2 to 10.0×10 -2  mol/l of Evans Blue or Trypan Blue as a first dye with about 0.5 to 2.0 volumes of about 0.2 to 10.0×10 -2  mol/l of Phloxine as a second dye; adjusting a pH of the system to 5.7 to 7.9 using 1/30 to 1/5 mols/l of a solvent selected from the group consisting of a phosphate buffer, a succinate buffer and a tris-acid buffer; and further adding a fixative agent selected from the group consisting of glutaraldehyde, formaldehyde and paraformaldehyde to the mixture in a concentration of 0.02 to 5.0%. 
     
     
       18. A dyeing agent according to claim 17, further comprising a surface active agent. 
     
     
       19. A dyeing agent according to claim 17, wherein upon mixing the first and second dyes, the first and second dyes cause no agglutination or sedimentation of the first and second dyes, and do not precipitate or agglutinate sugars, proteins or glycoproteins dissolved in said biological sample;   wherein the mixed first and second dyes are suitable for staining at least two objects to different respective degrees in color hue or in dyeing strength depending on the respective objects; and   wherein the mixed first and second dyes are suitable for staining respective components of a single object to different degrees in color hue or in dyeing strength.   
     
     
       20. A dyeing agent according to claim 17, wherein the dyeing agent is further obtained by mixing with the mixture one member selected from the group consisting of sodium azide, para-hydroxyphenylacetic acid, dehydroacetic acid and ethylenediaminetetraacetic acid in a concentration of 0.01 to 1.0%. 
     
     
       21. A dyeing agent for staining a biological sample, obtained by mixing one volume of about 0.2 to 10.0×10 -2  mol/l of Evans Blue or Trypan Blue as a first dye with about 0.5 to 2.0 volumes of about 0.2 to 10.0×10 -2  mol/l of Eosine as a second dye; adjusting a pH of the system to 5.7 to 7.9 using 1/30 to 1/5 mols/l of a solvent selected from the group consisting of a phosphate buffer, a succinate buffer and a tris-acid buffer; and further adding a fixative agent selected from the group consisting of glutaraldehyde, formaldehyde and paraformaldehyde to the mixture in a concentration of 0.02 to 5.0%, and sodium dodecyl sulfate as a surface active agent in a concentration of 0.01 to 0.5%. 
     
     
       22. A dyeing agent according to claim 21, wherein upon mixing the first and second dyes, the first and second dyes cause no agglutination or sedimentation of the first and second dyes and do not precipitate or agglutinate sugars, proteins or glycoproteins dissolved in said biological sample;   wherein the mixed first and second dyes are suitable for staining at least two objects to different respective degrees in color hue or in dyeing strength depending on the respective objects; and   wherein the mixed first and second dyes are suitable for staining respective components of a single object to different degrees in color hue or in dyeing strength.   
     
     
       23. A dyeing agent according to claim 21, wherein the dyeing agent is further obtained by mixing with the mixture one member selected from the group consisting of sodium azide, para-hydroxyphenylacetic acid, dehydroacetic acid and ethylenediaminetetraacetic acid in a concentration of 0.01 to 1.0%.